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Reconstructing the genetic history of Italians: new insights from a male (Y-
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Reconstructing the genetic history of Italians: new

insights from a male (Y-chromosome) perspective

Viola Grugni, Alessandro Raveane, Francesca Mattioli, Vincenza Battaglia,

Cinzia Sala, Daniela Toniolo, Luca Ferretti, Rita Gardella, Alessandro Achilli,
Anna Olivieri, Antonio Torroni, Giuseppe Passarino & Ornella Semino

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Torroni, Giuseppe Passarino & Ornella Semino (2018) Reconstructing the genetic history of
Italians: new insights from a male (Y-chromosome) perspective, Annals of Human Biology, 45:1,
44-56, DOI: 10.1080/03014460.2017.1409801

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ANNALS OF HUMAN BIOLOGY, 2018
VOL. 45, NO. 1, 44–56
https://doi.org/10.1080/03014460.2017.1409801

RESEARCH PAPER

Reconstructing the genetic history of Italians: new insights from a male

(Y-chromosome) perspective

Viola Grugnia, Alessandro Raveanea, Francesca Mattiolia, Vincenza Battagliaa, Cinzia Salab, Daniela Toniolob,
Luca Ferrettia, Rita Gardellac, Alessandro Achillia, Anna Olivieria, Antonio Torronia, Giuseppe Passarinod and
Ornella Seminoa
a
Dipartimento di Biologia e Biotecnologie “L. Spallanzani”, Universit
a di Pavia, Pavia, Italy; bDivisione di Genetica e Biologia Cellulare, Istituto
Scientifico San Raffaele, Milano, Italy; cDipartimento di Medicina Molecolare e Traslazionale, Universit
a di Brescia, Brescia, Italy;
d
Dipartimento di Biologia, Ecologia e Scienze della Terra, Universit
a della Calabria, Arcavacata di Rende, Cosenza, Italy

ABSTRACT ARTICLE HISTORY

Background: Due to its central and strategic position in Europe and in the Mediterranean Basin, the Received 31 July 2017
Italian Peninsula played a pivotal role in the first peopling of the European continent and has been a Revised 17 November 2017
crossroad of peoples and cultures since then. Accepted 20 November 2017
Aim: This study aims to gain more information on the genetic structure of modern Italian populations
and to shed light on the migration/expansion events that led to their formation. KEYWORDS
Subjects and methods: High resolution Y-chromosome variation analysis in 817 unrelated males from Y-chromosome variation;
10 informative areas of Italy was performed. Haplogroup frequencies and microsatellite haplotypes haplogroups; genetic
were used, together with available data from the literature, to evaluate Mediterranean and European history of Italy; Italian
inputs and date their arrivals. population
Results: Fifty-three distinct Y-chromosome lineages were identified. Their distribution is in general
agreement with geography, southern populations being more differentiated than northern ones.
Conclusions: A complex genetic structure reflecting the multifaceted peopling pattern of the
Peninsula emerged: southern populations show high similarity with those from the Middle East and
Southern Balkans, while those from Northern Italy are close to populations of North-Western Europe
and the Northern Balkans. Interestingly, the population of Volterra, an ancient town of Etruscan origin
in Tuscany, displays a unique Y-chromosomal genetic structure.

Introduction mainly spread in the Tuscan-Emilian Apennine area during

Due to its central and strategic position in Europe and in the
Mediterranean Sea, the Italian Peninsula played a pivotal role
in the first peopling of the continent and was, for millennia,
a crossroads of peoples, cultures, trades and languages (for
details, see Supplementary File S1).
Anatomically modern humans have inhabited the Italian
territory since the Upper Palaeolithic, as attested by archaeo-
logical and anthropological remains in caves like the Grotta
del Cavallo (Horse Cave) in Apulia, among which the oldest
(dated 45–43 thousand years ago - kya) European skeletal
remains were found (Benazzi et al., 2011). During the Last
Glacial Maximum (LGM), 25–19 kya, when large parts of
Europe were covered with thick ice sheets, Italy and the
Balkans were partly forested and probably acted as refuge
for Europeans escaping from the North. Traces of Neolithic
migratory events are spread all over Italy, with substantial
differences (especially between North and South) in the cer-
amics industry and archaeological remains. Signs of post-
Neolithic settlements are present in different areas of the
Italian territory, for example, the Nuraghe stones in Sardinia,
dated to the early Bronze Age, and the Villanovan culture,
the Iron Age.
A better knowledge of the genetic structure of the mod-
ern Italian population in the wider context of the European
continent and surrounding areas is, therefore, an essential
element to further reconstruct its genetic history and the
impact of ancient and more recent migration events.
Genetic variation of Italy
The first attempts to interpret the pattern of genetic variation
of the Italian Peninsula were made with classical polymor-
phisms, including the ABO blood group. Piazza et al. (1988)
studied the frequencies of 34 ‘independent’ alleles at seven
loci (ABO, MNS, KELL, RH, HP, HLA-A and HLA-B) and their
Principal Component (PC) analyses and synthetic maps of the
first three PCs are generally considered the foundations for
the Italian ‘genetic history’ reconstruction. These initial results
revealed a very distinct pattern for Sardinia, which is located
far away from all other Italian regions and represents an out-
lier in the European genetic landscape. When Sardinia was
excluded from the PC analysis, the first principal component

CONTACT Ornella Semino ornella.semino@unipv.it Dipartimento di Biologia e Biotecnologie “L. Spallanzani”, Universit
a di Pavia, Pavia, Italy

Subject classification codes: BG.24 Evolution and Population Genetics.
Supplemental data for this article can be accessed here.
ß 2017 Informa UK Limited, trading as Taylor & Francis Group

synthetic map, accounting for 27% of the total variation,
highlighted a North to South gradient, with similarities
between Northern Italy and Central Europe, in contrast to
the affinities between Central and Southern Italy with Greece
and other Mediterranean populations.
Later on, numerous studies introduced uniparental
markers to investigate the genetic structure of modern Italian
populations from either the maternal or paternal side
(Barbujani et al., 1995; Capelli et al., 2007; Destro Bisol et al.,
2008; Di Giacomo et al., 2003) and, in some cases, compara-
tively (Boattini et al., 2013; Brisighelli et al., 2012a, 2012b).
Overall, these studies suggested that the genetic structure of
modern Italy reflects, at least in part, the ethnic stratification
of pre-Roman times.
From the male perspective, most of the Italian Y-chromo-
some gene pool can be related to five main haplogroups:
R1b-M269, J2-M172, I-M170, G-M201 and E1b-M78. R1b is
more frequent in Northern Italy, while E1b, G and J2 harbour
higher frequencies in the South, suggesting a greater affinity
to West Europe for G and to South-East and South-Central
Europe for J2. Indeed, in the European context, the Italian Y-
chromosome variation (Capelli et al., 2007) fits the South/
East-North/West cline described in previous studies, which
was ascribed to the genetic admixture between incoming
Near Eastern farmers and pre-existing Mesolithic hunter-gath-
erers (Rosser et al., 2000; Semino et al., 1996, 2000, 2004). On
the other hand, the identification of an Anatolian (Asia
Minor) input in most of the Italian samples underlined that
pre-Neolithic populations were not completely replaced
(Capelli et al., 2007).
Unlike the majority of authors, Boattini et al. (2013) inter-
preted the Italian distribution of Y-chromosome genetic
diversity as non-clinal, but structured into three main areas
according to latitude: North-West Italy (NWI), South-East Italy
(SEI) and Sardinia. The outlier position of Sardinia was attrib-
uted to the extremely high frequency of I2-M26, a sub-
branch of haplogroup I also observed in the Iberian
Peninsula and Great Britain, but virtually absent in continen-
tal Italy, a finding clearly indicating a major founder event in
the Mediterranean island. The common genetic background
of Southern Italy and the Adriatic coast and the discontinuity
with Northern Italy and Tuscany were explained as due to a
“belt” area, where haplogroup frequencies changed more
rapidly than in other territories, possibly dating back to the
Neolithic when two independent and parallel diffusion proc-
esses occurred along the Adriatic and Tyrrhenian coastlines.
Our study provides novel Y-chromosome data, at a high-
resolution level, and extends comparative analyses to obtain
new insights concerning the genetic history of Italy and the
ancestral sources of the Italian gene pool.
Materials and methods
The sample
A total of 817 Italian individuals were newly-analysed in the
present study: 290 from Northern Italy (85 from the Borbera
Valley, an isolated valley of the Ligurian Apennine, at the
border between Piedmont and Liguria; 48 from the territories
ANNALS OF HUMAN BIOLOGY 45
of Voghera and Tortona, two towns at the border between
Pavia and Alessandria provinces; 157 from the Bergamo prov-
ince—78 from isolated valleys and 79 from the plain area of
the province), 113 from Central Italy (Volterra, a town in the
core area of ancient Etruria), 350 from Southern Italy (82
from Grec
ıa Salentina, 102 Apulians, 93 Calabrians from the
Ionian Coast, 73 Calabrians from the Tyrrhenian Coast) and
64 from Sicily. The sampling areas are shown in Figure 1;
geographic and historical information on the areas is pro-
vided in Supplementary File S1.
Sampled individuals were unrelated males with the pater-
nal grandfather born in the collection area and/or carrying a
monophyletic surname with a geographical distribution within
the sampling area as previously described (Zei et al., 2003).
Ethics statement
This research has been approved by the Ethics Committee
for Clinical Experimentation of the University of Pavia, Board
minutes of 5 October 2010. Geographical and ethnological
information such as ethnicity, language and genealogy were
ascertained by interview of blood donors after obtaining
their written informed consent.
DNA extraction
All DNAs were obtained from blood samples according to
standard phenol/chloroform extraction procedures, followed
by ethanol precipitation.
SNP genotyping
A total of 58 Y-chromosome biallelic markers were analysed
in a hierarchical way (Supplementary Table S1). They were
genotyped by AFLP, RFLP, DHPLC and direct sequencing after
PCR amplification of pertinent fragments.
The nomenclature used for haplogroup labelling is in
agreement with YCC conventions (The Y Chromosome
Consortium, 2002) and subsequent updates (Karafet et al.,
2008; King et al., 2011; Myres et al., 2011; Underhill et al.,
2010; van Oven et al., 2013).
STR genotyping
For each population, a sub-set of Y-chromosomes belonging
to the most representative haplogroups was analysed for the
microsatellite loci DYS19, DYS389I/II, DYS390, DYS391,
DYS392 and DYS393 (Kayser et al., 1997) in multiplex reac-
tions according to STRBase information (www.cstl.nist.gov/
biotech/strbase/y20prim.htm). The resulting STR haplotypes,
together with those available in the literature
(Supplementary Table S2), were used to investigate variation
and origins of haplogroups.
Statistical analyses
Y-chromosome haplotype and haplogroup variability of the
newly-analysed populations have been considered in the
46 V. GRUGNI ET AL.
Figure 1. Geographical locations of the 10 analysed Italian population samples.
wider Italian and European/Mediterranean context, together
with extended reference data available from the literature
(see Supplementary Table S3).
Haplogroup diversity was computed using the standard
method of Nei (1987). Comparison between groups was per-
formed using the Chi Square Test of independence (XLStat).
Genetic structure was examined through the Analysis of
Molecular Variance (AMOVA) (Excoffier et al., 1992) using the
Arlequin software Ver 3.5 and adopting different geographic
grouping criteria. Principal Components Analysis (PCA) on
haplogroup frequencies (Supplementary Tables S4 and S5)
was performed with XlStat, an Excel add-in, disregarding fre-
quencies below 5%. Median-Joining (MJ) networks (Bandelt
et al., 1999) were constructed using the Network 4.6.0.0 pro-
gram (Fluxus Engineering, http://www.fluxus.engineering.
com), after data processing with the reduced-median method
(Bandelt et al., 1995) and weighting of STR loci proportionally
to the inverse of the repeat variance. Geographical represen-
tations of the haplogroup frequency and mean STR variance
distributions were obtained with Surfer 6.0 (Golden Software)
following the Kriging procedure, as previously described
(Battaglia et al., 2009). Maps of microsatellite variances were
obtained after pooling data for locations with less than five
entries and assigning the resulting values to the centroid of
the pooled locations. Haplogroup ages were evaluated on
STR variation using the method proposed by Zhivotovsky
et al. (2004) and modified according to Sengupta et al.
(2006). A microsatellite evolutionary effective mutation rate
of 6.9  104 per generation (25 years) was used (Zhivotovsky
et al., 2004) since it is suitable in situations where the
elapsed time frame is >1000 years or 40 generations
(Zhivotovsky et al., 2006), like the pre-historic time depths
explored in this study. However, it is worth mentioning that
ambiguities related to past episodes of population history
(e.g. size fluctuations, bottlenecks, etc.) create inherent uncer-
tainties in the calibration of the Y-STR molecular clock, thus
age estimates of microsatellite variation should be consid-
ered with caution.
Results and discussion
Y-chromosome haplogroups in Italian populations
A total of 53 distinct lineages were identified; their frequency
distributions in the examined Italian populations are reported
in Supplementary Table S6 and illustrated according to their
phylogenetic relationships in Figure 2.
Haplogroup R is the most frequent (50.1%) with its two
main branches, R1a (4.7%) and R1b (45.3%), the latter mainly
accounted for by R1b-U152 (49.5% of the total R1b); R2 was

Figure 2. Haplogroup frequencies, as percentages, in the 10 analysed Italian population samples. BGV: Bergamo Valleys; BGP: Bergamo Plain; TO-VO: Tortona-
Voghera; VB: Borbera Valley; VOL: Volterra; AP: Apulia; GS: Grec
ıa Salentina; CAL-I: Ionian Calabria; CAL-T: Tyrrhenian Calabria; SIC: Sicily.
not observed. Next is haplogroup J (19.2%), mostly observed
as J2 (17.6%), and third is haplogroup E, as E1b (14.6%),
mostly represented by its ‘Balkan’ sub-clade E1b-V13. The
other main haplogroups show frequencies lower than 10%:
haplogroup G (8.4%) and haplogroup I (4.8%).
The frequency distributions of the three main haplogroups
(R, J and E) and those of their sub-clades in Italian popula-
tions are illustrated in Figure 3. The black sectors in the pri-
mary pies are proportional to the frequency of the main
haplogroup in the different populations, whereas the col-
oured sectors in the secondary pies are proportional to the
frequencies of the different sub-clades within the relative
main haplogroup. These haplogroups, which account for
more than 80% of our Italian samples, are also highly repre-
sented in Europe (Chiaroni et al., 2009). Frequency and vari-
ance distribution maps (Supplementary Figures S1 and S2)
were obtained for their most informative sub-haplogroups.
Networks of the associated STR-haplotypes were also con-
structed (Supplementary Figures S1–S3) and relative coales-
cent ages were estimated for each population/area
(Supplementary Tables S7 and S8).
ANNALS OF HUMAN BIOLOGY 47
Northern Italy (Bergamo Valleys and plain, Tortona-
Voghera and Borbera Valley) is characterised by an extremely
high incidence of the R1b haplogroup (69.0%) when com-
pared to all the other main haplogroups whose frequencies
do not reach 10%. This haplogroup, which characterises a
wide portion of the gene pool of the examined populations,
shows a decreasing frequency pattern from North to South
Italy, where it shows its lowest incidence (27.5%). This pat-
tern is virtually totally ascribable to R1b-U152, the most rep-
resented R1b sub-lineage, whereas no frequency gradients
were detected for the other sub-lineages. R1b-S116
(xU152,
M529) is equally represented in all the Italian populations
(Figure 3, dusty rose sector in secondary pies). This shows
the highest frequencies in two isolated areas of Northern
Italy: Borbera Valley (12.9%) and Bergamo Valleys (17.9%).
The frequency peak is particularly noticeable in Bergamo
Valleys in comparison to the neighbouring plain area (17.9%
vs 3.8%, respectively, p < .01). Global frequency and variance
distributions of haplogroups R1b-U152 (not shown) and R1b-
S116
(Supplementary Figure S1) are coherent with a North
and West European origin, respectively. Network analyses
48 V. GRUGNI ET AL.
Figure 3. Frequencies of the main Y-chromosome haplogroups E1b, J2 and R1b and their sub-clades in the 10 analysed Italian population samples. Black sectors in
the primary pies are proportional to the frequency of the main haplogroup in each population. Coloured sectors in the secondary pies are proportional to the fre-
quencies of sub-haplogroups within the relative main haplogroup.
reveal high internal complexities, especially for R1b-U152
(Supplementary Figure S3), due to unequal contributions of
different sub-lineages, as previously noted by Valverde et al.
(2016) in Spain for R1b-S116
(xU152, M529) and by Boattini
et al. (2013) and the FamilyTree dataset (http://www.davidk-
faux.org/R1b1c10_Resources.pdf) for R1b-U152. However,
both networks are characterised by a main demographic
expansion centred in North-West and Central-North Europe,
which strongly affected Northern Italy. Thus, taking into
account that the highest reported incidence of R1b-
S116
(xU152, M529) is in Iberia (Adams et al., 2008; Myres
et al., 2011), its high frequency in the relatively isolated pop-
ulations of the Bergamo and Borbera Valleys could represent
the outcome of ancient gene flow from that area, possibly
magnified by genetic drift. On the other hand, R1b-M412
,
so far described only in Turkey, Iran, Cyprus and Crete (Myres
et al., 2011; Voskarides et al., 2016), is observed in all the
four Southern Italian samples, all from the ancient Magna
Graecia area, but only sporadically in population groups from
Northern Italy. The R1b-M412
Y chromosomes could, there-
fore, represent the legacy of an Eastern Mediterranean input
associated with the early Hellenic colonisation, and/or the
more recent Byzantine domination. This scenario is supported
by the high frequency of R1b-M412
in the Griko-speaking
community of Grec
ıa Salentina (13.4%), where haplogroup
R1b-M412
probably reflects ancient colonisation events
from Greek-speaking islands rather than continental Greece.
The R1a haplogroup is observed along the entire
Peninsula. With the exception of the Tortona-Voghera sam-
ple, it displays lower frequencies in the North and in the
Centre in comparison with the southern populations, espe-
cially those of the Ionian Coast (8.6% in Ionian Calabria, 5.9%
in Apulia and 15.8% in Grec
ıa Salentina). R1a-M17 represents
an important component of the modern gene pool of
Greece, where it reaches its highest frequencies (16.3% and
22.0%, in mainland Greece and in Thracia, respectively)
(Battaglia et al., 2009; Heraclides et al., 2017). Taking into
account that it is found virtually only as R1a-M17
(xM458) in
both the Southern Italian samples and in mainland Greece, it
is likely that R1a-M17
is a signature of the Southern Balkan
(mainland Greece) influence into Southern Italy. Thus, differ-
ently to haplogroup R1b-M412
, R1a-M17
seems a hallmark
of a significant male seaborne input from Balkan populations
towards the eastern coast of Southern Italy.
Unlike R1b, haplogroup J frequency increases from North
(8.3%) towards Central (13.3%) and Southern Italy, where it
reaches the highest value (28.5%). The distribution of hap-
logroup J1 is restricted to South Italy; this haplogroup, which
arose in the southern part of the Middle East (Malaspina
et al., 2000; Nebel et al., 2002; Semino et al., 2004), character-
ises Near Eastern and North African Arabic-speaking popula-
tions (Al-Zahery et al., 2011; Chiaroni et al., 2009; Heraclides
et al., 2017; Tofanelli et al., 2009). Its presence in the south-
ern part of the Italian Peninsula indicates gene flow from
these populations. In contrast, haplogroup J2, which most
likely arose in the northern part of the Middle East (King
et al., 2008; Malaspina et al., 2000; Nebel et al., 2002; Semino
et al., 2004) and spread in association to Neolithic and post-
Neolithic migrations, is present all over the Italian territory,
although with relevant differences in the distributions,
probably due to more recent migratory events. J2a is most
represented in southern populations, where its J2a-M530 and
J2a-Page55
sub-lineages (with a remarkable frequency of
the latter in Apulia) were the most prevalent. Its lowest fre-
quencies were registered in the isolated populations of
Borbera Valley (3.5%, only represented by J2a-M530) and
Bergamo Valleys, where this clade was not observed at all.
J2a-M67
, which is widely distributed in Europe, the Middle
East and North Africa (Supplementary Figure S2), with a not-
able peak in Portugal, is mainly present in South Italy, espe-
cially in Ionian Calabria. Its variance map shows instead a
different pattern, with high values in some Middle Eastern

regions, such as Iran, Turkey and Palestine, but also in the
two main islands of the eastern Mediterranean Sea as well as
along the other Mediterranean coastal regions. These data
indicate that this haplogroup might have spread by sea,
probably starting from the Middle East. The network analysis
reveals a complex internal heterogeneity as well as an expan-
sion that affected not only Middle Eastern populations but
also the Balkans and Southern Italy. The most frequent
haplotype in the network comprises subjects mainly from
Middle Eastern populations, including Crete and Cyprus, but
also from Southern Italy. Notably, Northern Italians are not
present in the central haplotype, suggesting a possible later
arrival to this area. The great majority of the Portuguese Y
chromosomes belong to only one haplotype, thus revealing
a very recent expansion of this lineage in western Iberia. The
oldest ages based on microsatellite variation are in Cyprus
(16.6 ± 4.7 kya), Crete (16.3 ± 5.5 kya) and Apulia (16.6 ± 7.0
kya), followed by those in the Middle Eastern populations.
Notably, North Sardinia, Tuscany and Sicily also have high
coalescent times (Supplementary Table S7). These data sug-
gest an overall diffusion of J2a-M67
both by sea and by
land. The finding of such high variance and coalescence
times in the two main islands of the Aegean Sea, character-
ised by an early spread of agriculture, is in agreement with
the scenario that the first steps of the Neolithic spread were
indeed towards Cyprus and Crete (King et al., 2008). On the
other hand, diffusion by land from the Middle East towards
the Southern Balkans is less likely. The high variance and
coalescent time values observed in Southern Italian regions,
overall comparable to those of the Middle East, can be the
outcome of seaborne migrations from different geographic
sources at different times. For example it is known that, in
post-Neolithic times 5 kya, North West Anatolia developed
a complex society engaged in a widespread Aegean trade
referred to as “Maritime Trojan culture”, involving both the
Western Anatolian mainland and several large islands in the
Eastern Aegean Sea (Korfmann, 1997). Interestingly, J2a-M67

also harbours a high microsatellite variation age in Volterra,
which is located in the core area of ancient Etruria. Multiple
hypotheses have been proposed concerning the origin of
Etruscans, but our observations tend to support the view
that Asia Minor was the ancestral source of the Etruscan
gene pool, as already proposed by Achilli et al. (2007) on the
basis of mtDNA data.
J2a-M92 is widely distributed in the Middle East, the
Balkans, and along the Mediterranean coast. In Italy, it shows
a high frequency in the South, especially in the Southern
part of Apulia. The variance map shows peaks in Turkey and
Sicily, followed by the Southern Balkans. The highest ages
based on microsatellite variation are observed in Sicily
(11.8 ± 3.4 kya) and Turkey (11.7 ± 4.9 kya). Since the fre-
quency and variance maps suggest a possible origin of J2a-
M92 in and around Turkey, the observation of an age in
Sicily that is close to that observed in Turkey may indicate an
ancient migration from Turkey to Sicily. Comparable high
coalescent times based on microsatellite variation are
observed in Greece (7.2 ± 2.0 kya), Apulia (7.3 ± 3.1 kya) and
Tuscany (8.7 ± 2.7 kya). These data most likely testify sea-
borne contacts of these Italian regions with Eastern
ANNALS OF HUMAN BIOLOGY 49
Mediterranean Neolithic civilisations, although the stratifica-
tion of different migratory events could also have contrib-
uted to the internal heterogeneity, especially in Tuscany.
J2b is most frequent in the Tortona-Voghera sample,
which is located in the open Po Valley, and in Apulia, which
faces the Adriatic Sea, while it is present at low frequencies
in the Tyrrhenian sample of Calabria and not observed in
Sicily. Interestingly, its incidence in the Volterra sample is
comparable to that observed along the Salentina Coast and,
as in the northern samples, it is mainly represented by the
“Balkan” J2b-M241.
On the whole, the heterogeneous distribution of J2 sub-
haplogroups in modern Italians highlights different diffusion
routes. Sub-haplogroups J2a-M530 and J2a-Page55
, as J2a-
M67
, probably mark gene flow events from the Middle East
(they display the highest frequency and variances in Iran
(Grugni et al., 2012)) across the Caucasus, Turkey (data not
shown), Cyprus (Voskarides et al., 2016) and Crete (King
et al., 2011) towards Southern Europe, that affected mainly
the southern regions of Italy. Differently, J2b-M241 which dis-
plays a strong expansion in the Southern Balkans (Battaglia
et al., 2009; Cruciani et al., 2007; Karachanak et al., 2013) and
has been associated with Neolithic and post-Neolithic migra-
tions from Greece and the Balkans (Battaglia et al., 2009;
King et al., 2008), marks a seaborne route whose contribution
is still detectable along the Adriatic coast (Boattini et al.,
2013) as well as in populations along the Po Valley. It is
worth underlining the presence of sub-haplogroup J2a-M92
at 7.3% in Grec
ıa Salentina. This value, comparable with that
observed in the sample from Lecce (Boattini et al., 2013), is
suggestive of a direct, or Balkan-mediated, seaborne contri-
bution from Asia Minor (Grugni et al., 2012).
Haplogroup E, mostly represented by E1b-M78, increases
in frequency from North (8.3%) to South, where it reaches an
incidence of 21.3%. Its main sub-clade, E1b-V13, displays a
decreasing frequency cline from the Southern Balkans to
Western Europe (Supplementary Figure S2) and is also pre-
sent, at lower frequencies, in Anatolia and all along the
Italian Peninsula. Similar to J2b-M241, the E1b-V13 sub-clade,
which spread from the Balkans (Battaglia et al., 2009;
Cruciani et al., 2007; Karachanak et al., 2013), is mainly
observed in the South of Italy, with frequencies higher than
10% in Apulia; however, unlike the Balkan J2 branch, it is
also found in Sicily. The distribution of its variance
(Supplementary Figure S2) parallels the frequency clinal pat-
tern, although high variance values are also observed in
Central-Eastern Europe and a major peak is present in
Anatolia. In Italy, the variance is highest in the South. The
highest microsatellite age estimates (Supplementary Table
S7) are in Turkey (10.0 ± 3.4 kya), where this clade likely origi-
nated (Battaglia et al., 2009; Cruciani et al., 2007; Karachanak
et al., 2013). Indeed, the variance is also highest in the same
areas. The archaeological congruence between the Greek and
Southern Anatolia Mesolithic may explain the similar E1b-V13
expansion times (Perl
es, 2001). In Italy, E-V13 shows coales-
cent age and variance values similar to the Northern Balkan
ones. These data are in agreement with a first migration of
E1b-V13 from Anatolia towards the Southern Balkans, where
it underwent a demographic expansion, followed by a later
50 V. GRUGNI ET AL.
spread towards Southern Italy (Battaglia et al., 2009). The
relatively recent expansion times in the Balkans are consist-
ent with the Balkan Bronze Age, a period that saw strong
demographic changes as demonstrated by archaeological
records (Childe, 2013; Kristiansen, 2000), and could therefore,
represent a possible time frame for the population move-
ment into the South of Italy.
E1b-V13 is also observed in Volterra and the Northern
Italian groups, mainly in the most accessible areas (Boattini
et al., 2013). This observation supports a Balkan influence in
Northern Italian populations as well, most likely through an
Adriatic route and along the Po Valley and, to a lesser extent
in lateral, more isolated, mountainous valleys. Among the
other E sub-clades, the Middle Eastern E1b-M34 lineage is
restricted to Apulia, Calabria and Sicily, whereas the North
African E1b-M81, E1b-V22 and E1b-M35
are observed in
Calabria and Sicily. In particular, E1b-M81, which is very fre-
quent in North Africa, reaches an incidence of 6.3% in Sicily.
This marker has also been observed at significant frequencies
in Southern Iberia and its presence in Southern Europe has
been attributed, due to its low microsatellite variation, to
relatively recent migration(s) from North Africa (Adams et al.,
2008; Di Gaetano et al., 2009; Flores et al., 2003, 2004;
Semino et al., 2004). On the other hand, the finding of E1b-
M35
Y chromosomes (3.5%) in the Borbera Valley is not
completely unexpected. Indeed, the so called “Vie del Sale -
Salt Paths” in the high Valley could have been the entry
route of North-East African Y chromosomes either during the
passage of the Attila Army in the 5th century and/or the
numerous Saracen invasions around the year 1000 A.D.
Haplogroup G is not characterised by a clinal distribution
pattern. Frequencies higher than 10% were registered in the
Borbera Valley (15.3%), Volterra (13.3%), Tyrrhenian Calabria
(12.3%) and Apulia (11.8%). Interestingly, most of the samples
belong to the sub-haplogroup G2a-L497, which, as R1b-U152,
displays a pattern of expansion from Central-North Europe
(Supplementary Figure S1). Its influence in Italy is mainly
appreciable in the Borbera Valley, where it accounts for more
than 80% of haplogroup G, and in Volterra. On the other
hand, almost all the G clades seem to be present in Southern
Italy. The G2a-L91 branch, common among Anatolian farmers
of 8 kya (Lazaridis et al., 2016; Mathieson et al., 2017) and
€
characterising the Otzi’s Y chromosome with its sub-clade
G2a-L166, was observed in Tyrol (Berger et al., 2013), Tuscany
(Francalacci et al., 2013) and in a great portion of the
Southern Corsican (25%) and North Sardinian (9%) Y-chromo-
some gene pools (Keller et al., 2012). In this study, it was
recorded in one subject from the Borbera Valley and one
from Apulia, indicating that, although rare, the G2a-L91 hap-
logroup is present in the entire Peninsula.
The frequency of haplogroup I is similar in North and
South Italy (4.8% and 4.1%, respectively) and higher in the
Centre (7.1%), with most of the samples belonging to the
I1-M253 and I2-M223 lineages. The detection of I2-M26 Y
chromosomes in Volterra is noteworthy. This sub-haplogroup
is known for its high frequency (>30%) in Sardinia
(Francalacci et al., 2013; Rootsi et al., 2004; Zei et al., 2003),
so its presence in the Volterra sample suggests a connection
between Tuscany and Sardinia. This link could be related to
Table 1. Gene diversity in Italian populations.
BGV BGP TO-VO VB VOL AP GS CAL-I CAL-T SIC
0.774 0.701 0.861 0.865 0.916 0.948 0.933 0.960 0.960 0.959
BGV: Bergamo Valleys; BGP: Bergamo Plain; TO-VO: Tortona-Voghera; VB:
Borbera Valley; VOL: Volterra; AP: Apulia; GS: Grec
ıa Salentina; CAL-I: Ionian
Calabria; CAL-T: Tyrrhenian Calabria; SIC: Sicily.
the first peopling of the island or represent the signature of
the extensive trade exchanges that Etruscans had with
Sardinia, or alternatively could be due to the rather recent
migration of numerous shepherds from the island to
Tuscany. However, taking into account that (i) all the Volterra
I2-M26 Y chromosomes belong to the deepest and less rep-
resented branches (
-star- and alfa) of I2-M26 (data not
shown), not involved in the expansion of this clade in
Sardinia (Francalacci et al., 2013), (ii) the carriers of these
chromosomes belong to families that reside in Volterra from
at least four generations and (iii) all are characterised by local
monophyletic surnames, we can exclude that their presence
in Tuscany is due to recent gene flow.
Finally, haplogroup T, which arose and began to differenti-
ate in the Near East about 25 kya (Mendez et al., 2011) and
is observed at low frequencies in Europe and in parts of the
Middle East, North and East Africa (Heraclides et al., 2017),
could be a potentially informative marker to discriminate
movements in the Mediterranean area. In Italy, it displays fre-
quency spots in central and southern regions (Boattini et al.,
2013) and appears sporadically in the North-West; however,
the present level of resolution does not provide any useful
information to better understand its diffusion.
Gene Diversity values based on haplogroup frequencies
for each of the analysed Italian populations are reported in
Table 1.
Lower indexes characterise northern populations com-
pared to southern ones, indicating a minor Y-chromosome
haplogroup variation in the North. The lowest values were
observed in the Bergamo populations followed by those in
Tortona-Voghera and Borbera Valley, whereas the highest
ones were in Calabria (with no differences among the two
sub-sets) and Sicily, followed by Apulia and Grec
ıa Salentina.
This high Y-chromosome diversity characterising Southern
Italian populations is in agreement with the complex and
extensive patterns of pre- and proto-historical admixture
recently reported for the populations of this area on the
basis of genome-wide data (Sarno et al., 2017).
The Italian Y-chromosome gene pool in the European
and Mediterranean contexts
In order to visualise the relationships between the analysed
groups with other Italian populations and place them in a
wider European and Mediterranean context, PCAs on hap-
logroup frequencies were carried out exploiting available
data from the literature normalised to the highest possible
level of phylogenetic resolution.
A first PCA was performed by using the largest dataset
taken from the literature (Supplementary Table S4), but at a
low level of haplogroup resolution, in order to compare the
 ANNALS OF HUMAN BIOLOGY 51

Figure 4. Principal Components (PC) plot based on the frequencies of low-resolution Y-chromosome haplogroups (Supplementary Table S4). Numbers in parenthe-
ses indicate the proportion of the total genetic information retained by a given PC. The inset plot illustrates the contribution of each haplogroup. The three non-
Italian groupings (Middle East-Asia Minor-Caucasus, West Europe, East Europe) are circled. AG: Agrigento; AMA: Appenine Marche; AP: Apulia; AQ: L’Aquila; Bas:
Baschi; BGP: Bergamo Plain; BGV: Bergamo Valleys; BN: Benevento; BO: Bologna; BS: Brescia; Bulg: Bulgaria; CAL-I: Ionic Calabria; CAL-T: Tyrrhenian Calabria; Cau:
Caucasus; CCK: Cosenza/Catanzaro/Crotone; CE: Piceni; CE SARD: Central-East Sardinia; CMA: Central Marche; CN: Cuneo; CO: Como; CP: Campobasso; Cre: Crete; Cro:
Croatia; CS SARD: Central-South Sardinia; CT: Catania; CTU: Central Tuscany; ELB: Elba Island; ES: East Sicily; GR-SN: Grosseto/Siena; Gre: Greece; GS: Grec
ıa Salentina;
Iran: Iran; LE: Lecce; LIG: Liguria; Mac: Macedonia; MC: Macerata; MT: Matera; N SARD: North Sardinia; NEI: North-East Italy; NEL: North-East Latium; NWA: North-West
Apulia; PG: Foligno; PT: Pistoia; PUGR: Grecanici; RG-SR: Ragusa/Siracusa; RN: Rimini; SAN: Sanniti; SAP: South Apulia; Serb: Serbia; SIC: Sicily; SIC SW: South-West
Sicily; SLA: South Latium; SP-MS: La Spezia/Massa; SV-GE: Savona/Genova; SW: Belvedere; TLB: Tuscany Latium Border; TO-VO: Tortona-Voghera; Turk: Turkey; TV:
Treviso; UD: Udine; VB: Borbera Valley; VI: Vicenza; VLB: Badia Valley; VM: Valmarecchia; VOL: Volterra; WCL: West Calabria; WCP: West Campania; WS: West Sicily.

majority of the analysed samples. The plot of the first two
PCs, which together explain more than 29% of the variance,
is shown in Figure 4, together with an inset plot illustrating
haplogroup contributions.
The first PC (F1) which explains 16.22% of the total vari-
ance, clearly separates all the Middle East, Asia Minor and
Caucasus samples characterised by high frequencies of hap-
logroups E, G and J, from the European ones, showing a
high frequency of haplogroup R instead. The second PC (F2)
explains 13.70% of the total variance and distinguishes the
Middle East and Asia Minor populations from the Caucasus
groups based on different frequencies of haplogroups J and
G and Eastern from Western European groups based on
uneven R1a and R1b frequencies. Italian populations are dis-
tributed among these four groupings, but with a broad lati-
tudinal separation generated by the first PC, since some
populations from the South reside close to those of North
Italy and vice versa. This distribution, even if based on low-
resolution data, underlines the variegated pattern of genetic
variation in Italy and is rather informative in terms of past
migration inputs. For instance, the closeness of Southern
Italians to the Middle East is due to a high frequency of hap-
logroup J, which is typical of the Middle East and has been
associated with migrations from this area, including the
spread of agriculture by Middle Eastern farmers during the
Neolithic period. Therefore, the low genetic distance between
South Italian and Middle Eastern populations—and, con-
versely, the higher distance between Middle Eastern and
North Italian populations—can be explained by a greater
influence of Middle Eastern Neolithic farmers and post-
Neolithic migrants from Eastern Mediterranean populations
into the South rather than in the North of Italy. On the other
hand, Northern Italians show a general closeness to the
Basques, sharing a high incidence of the R1b clade.
Furthermore, the proximity of some North-Eastern Italian
groups to the Balkan cluster, mainly due to haplogroups R1a-
M17, E1b-M78 and I-M170, suggests a Balkan contribution.
This is the case, for example, of the Udine (UD) and Vicenza
(VI) samples. The separation of Treviso (TV) from the geo-
graphically close Vicenza, due to the second PC, reflects
instead the high incidence of the western R1b-M269 lineage
in the former. A major Balkan influence on Vicenza rather
than on Treviso could explain this observation, but it is worth
noting that both groups are rather small (VI ¼ 40 and
TV ¼ 33) and that the second PC encompasses only a part of
the genetic variance.
Although low-resolution haplogroups capture only some
of the total variability, regional differences are also identified
52 V. GRUGNI ET AL.

Figure 5. Principal Components (PC) plot based on the frequencies of high-resolution Y-chromosome haplogroups (Supplementary Table S5). Numbers in parenthe-
ses indicate the proportion of the total genetic information retained by a given PC. The inset plot illustrates the contribution of each haplogroup. The three non-
Italian groupings (Middle East-Asia Minor-Caucasus, West Europe, Balkans) are circled. Ala: Araba; AP: Apulia; Bba: Bizkaia; Bea: Bearn; BGP: Bergamo Plain; BGV:
Bergamo Valleys; Big: Bigorre; Boc: West Bizcaia; Bulg: Bulgaria; BUR: Burgos; CAL-I: Ionian Calabria; CAL-T: Tyrrhenian Calabria; Can: Cantabria; Cau: Caucasus; CE
SARD: Central-East Sardinia; Cha: Chalosse; Cre: Crete; Cro: Croatia; CS SARD: Central-South Sardinia; Gre: Greece; GS: Grec
ıa Salentina; Gso: South-West Gipuzkoa;
Gui: Gipuzkoa; Herz: Herzegovina; Mac: Macedonia; Nar: North Aragon; Nco: Central-West Nafarroa; Nla: Lapurdi Nafarroa; Nno: North-West Nafarroa; N SARD: North
Sardinia; Rio: La Rioja; Ron: Roncal: Salazar Valley; Serb: Serbia; SIC: Sicily; Sou: Zuberoa; TO-VO: Tortona-Voghera; Turk: Turkey; VB: Borbera Valley; VOL: Volterra;
Zmx: Lapurdi Baztan.

in other areas of the Peninsula. For instance, South Apulian
groups (GS, AP and LE) turned out to be far away from
North-Western Apulia (NWA) and located between the Near
Eastern and Balkan population clusters, a finding that is
strongly suggestive of a Greek influence, especially for the
Grec
ıa Salentina (GS). Conversely, the proximity of the “non-
Grecanic” Apulians (AP) to Crete, one of the first areas
reached by Neolithic Near Eastern farmers, supports the scen-
ario that the same genetic stock also reached Apulia by sea.
The relationships between the different Italian groups in
the general context of the European and Mediterranean pop-
ulations became much clearer when the PCAs were per-
formed at a higher level of haplogroup resolution. The plot
of the two PCs obtained from this analysis and a plot display-
ing the contribution of each haplogroup to the first and
second PC are shown in Figure 5.
On the whole, the first two PC plots explained 37.33% of
the total variance. The obtained distribution shows an overall
general agreement with geography: while the first compo-
nent accounts for a northwest–southeast separation, the
second component discriminates mainly according to lati-
tude. Thus, as in the previous analysis, the Basque groups are
at the opposite extreme of the first PC relative to the Middle
East, Turkey and the Caucasus cluster. Balkan populations are
sub-divided into two groups, one including Greece and
Bulgaria and the other encompassing all the populations
from the Northern Balkans and Macedonia. It is worth noting
that the Italian populations do not group together in this
analysis, but are scattered in the space comprising the
above-mentioned clusters. The first component separates
Bergamo Valleys (BGV) and Central-East Sardinia (CE SARD)
from all other Italian populations for their high frequency of
haplogroup R1b, especially R1b-S116
, whereas Tortona-
Voghera (TO-VO) and the Borbera Valley (VB) are closer to
the Balkans due to the high incidence of I2-M423, I2-M438
and E1b-M78, markers that are largely present in Eastern
Europe. Sardinian groups have been pulled down in the plot
by the high prevalence of I2-M26 marker, also observed at
low frequency in some Basque and Italian groups. Notably,
while Bergamo plain (BGP) appears more related to the
Eastern European populations, Bergamo Valley (BGV) is closer
to Basques. For a long time, Basque populations have been
considered the “living fossils” of the earliest modern
European inhabitants, both from genetic and linguistic points
of view (Cavalli-Sforza et al., 1994; Richards et al., 1996), but
more recent studies based on ancient genome-wide sequenc-
ing data suggest that they are the results of long-lasting iso-
lation of a population group originated by the admixture of
 ANNALS OF HUMAN BIOLOGY 53

local hunter-gatherers and early farmers (Gu €nther et al.,
2015). This might be interpreted as indicating that the valley
populations of the Bergamo province were more isolated
than those inhabiting the plain and, thus, they might have
better retained traces of the Y-chromosome gene pool of
Western European hunter-gatherers. Thus, the first compo-
nent indicates contiguity of the Y-chromosome haplogroup
composition between Basques, North Italy (in particular with
Bergamo Valleys) and Sardinia, especially the Central-East
area of the island, which is known as the “archaic zone”. The
Volterra sample (VOL) occupies an intermediate position,
between North and South. As already suggested by the low
resolution PCA (Figure 4), different gene flows affected
Southern Italian populations: Ionian Calabria (CAL-I) and
Grec
ıa Salentina (GS) are very close to Greece and Bulgaria,
whereas the other samples from Apulia (AP) are closer to
Crete. The differences among the Apulian populations are
explained by a dissimilar incidence of R1b-M412
and R1a-
M17, which are considerably higher in Grec
ıa Salentina than
in its neighbouring areas, and of G2a-P15, which is higher in
Apulia. A great difference is also observed among Calabrian
populations: the sample from the Ionian coast (CAL-I), due to
the higher incidences of E1b-M78 and R1a-M17, is closer to
the Southern Balkans than the one from the Tyrrhenian coast
(CAL-T). In the absence of data from African groups, individu-
als from Sicily (SIC) appear in the proximity of Central-South
Sardinia (CS-SARD), the only other Italian population showing
a considerable incidence of haplogroup E1b-M81. When a
PCA was performed at lower resolution (data not shown) in
order to include samples from North Africa, where hap-
logroup E1b-M81 reaches high frequencies, Sicily further sep-
arated from other Italian populations. On the whole, the
high-resolution PC analysis confirms a strong Middle Eastern
influence in Southern Italian populations, which show
high frequencies of haplogroups J2a and G2a (J2a-M410 and
G2a-P15).
To further investigate the significance of the Italian gen-
etic structure displayed by PCAs, we also carried out analyses
of molecular variance (AMOVA) on the population samples
from this study, as well as those from Boattini et al. (2013).
The results are summarised in Table 2.
Boattini et al. (2013) reported that North-West and South-
East Italy are not separated according to latitude, but by a
longitudinal line, and explained this difference by at least
two independent diffusion processes involving the western
and the eastern coasts of the Italian Peninsula during the
Neolithic revolution. To evaluate this scenario, we performed
first an AMOVA with both datasets and grouped the samples
according to the previously employed criteria (Boattini et al.,
2013). Specifically, three groups were considered: North-West,
South-East and Sardinia. In subsequent tests, alternative
groupings were investigated, but none produced better
results. For example, when Volterra, a potential derivative of
the ancient Etruscan people, was excluded, the variation
among groups decreased (from 10.62% to 9.32%), thus con-
firming the pattern of variation uncovered by Boattini et al.
(2013). However, this analysis was at a very low level of hap-
logroup resolution, which is inadequate for micro-geographic
analyses, thus further AMOVA tests were carried out on our
data at the maximum level of haplogroup resolution and the
population samples were assembled according to the high-
resolution PCA grouping (Figure 5). The results are reported
in Table 3.
The first analysis was performed by considering the fol-
lowing three population groups: North Italy, made up by all
North Italian samples; South-East Italy, which included Apulia,
Grec
ıa Salentina and Ionian Calabria; and South-West Italy,
which comprises Sicily and Tyrrhenian Calabria. The analysis
produced a value of variation among groups of 10.69%, simi-
lar to that (10.62%) obtained by using low-resolution hap-
logroups and the population sub-division proposed by
Boattini et al. (2013). A higher percentage of variation among
groups (11.68%) was obtained when the Borbera Valley and
Volterra samples were set apart; this could indicate that
these two populations differ from the others, likely represent-
ing genetically isolated groups. However, when only the

Table 2. AMOVA analysis with low-resolution haplogroups.

Sub-division criterion Source of variation Variance components Percentage of variation
North-West Italy vs South-East Italy vs Sardinia (3 groups) Among groups 0.39217

10.62
Among populations within groups 0.05303

1.44
Within populations 3.24769

87.94
North Italy vs South Italy vs Central Italy vs Sardinia (4 groups) Among groups 0.33912

9.32
Among populations within groups 0.05154

1.42
Within populations 3.24769

89.26


p < .01.

Table 3. AMOVA analysis with high-resolution haplogroups.

Sub-division criterion Source of variation Variance components Percentage of variation
North Italy vs South-East Italy vs South-West Italy (3 groups) Among groups 0.56305
10.69
Among populations within groups 0.10577

2.01
Within populations 4.60023

87.31
North Italy vs South Italy vs Borbera Valley vs Volterra (4 groups) Among groups 0.61477

11.68
Among populations within groups 0.04669
0.89
Within populations 4.60023

87.43
North Italy vs South Italy vs Volterra (3 groups) Among groups 0.65349
12.31
Among populations within groups 0.05669
1.07
Within populations 4.60023

86.63

p < .05;

p < .01.
54 V. GRUGNI ET AL.
Volterra sample was kept separate and the Borbera Valley
was pooled with the northern samples, the among-group
variation increased, reaching the highest value among
groups (12.31%), while the variance among populations
within groups remained almost the same. This suggests a
notable difference among the three groups and, in particular,
underlines a significantly different Y-chromosome haplogroup
composition between modern Volterra and the rest of Italy.
Since the population from Volterra could be, at least in part,
of Etruscan ancestry, it would be interesting to verify if other
populations from Tuscany (analysed at high-resolution level)
show the same peculiarity. If so, our observation would be in
line with the scenario of a foreign source for the ancient
Etruscan people, instead of an autochthonous origin.
Conclusions
In this paper, Y chromosomes of 817 subjects from inform-
ative areas of the Italian Peninsula were analysed at a high
level of haplogroup resolution. The results, compared with
those available from the literature, provided a more detailed
overview of the Y-chromosome variation in Italy relative to
previous studies. A genetic structure characterised by high
complexity emerged, probably reflecting the multifaceted
pattern of peopling of the Italian Peninsula. Specifically, the
southern groups are characterised by a higher haplogroup
variation in comparison with those from the North. This is
well illustrated by the AMOVA analysis with ‘high-resolution
’haplogroups, which provided the best results when three
groupings were considered: Northern Italy, Southern Italy
and Volterra sample. Thus, the population groups are better
separated according to their latitude rather than their longi-
tude, as proposed by Boattini et al. (2013), and also con-
firmed by our analysis at a comparable low-resolution level.
However, isolated populations such as those from the
Borbera Valley and Grec
ıa Salentina still preserve unique hap-
logroup distributions, due to either genetic drift and/or the
legacy of distinctive ancestral sources.
When compared to other populations, Italian samples do
not cluster all together, but are distributed among European
and Mediterranean people. Southern samples show a higher
similarity with Middle Eastern and Southern Balkan popula-
tions than northern ones; conversely, northern samples are
genetically closer to North-West Europe and Northern Balkan
groups. The intermediate position of Volterra, between South
and North Italy, is a mark of its unique Y-chromosomal gen-
etic structure. However, the long-lasting debate concerning
the origin of Etruscans remains open. As a matter of fact,
while the presence of J2a-M67
suggests contacts by sea
with Anatolian people, in agreement with the Herodotus
hypothesis of an external Anatolian source of Etruscans, the
finding of the Central European lineage G2a-L497 at consid-
erable frequency would rather support a Northern European
origin of Etruscans. On the other hand, the high incidence of
European R1b lineages cannot rule out the scenario of an
autochthonous process of formation of the Etruscan civilisa-
tion from the preceding Villanovan society, as first suggested
by Dionysius of Halicarnassus; a detailed analysis of
haplogroup R1b-U152 could prove very informative in this
regard.
Acknowledgements
The authors are grateful to all the donors for providing biological speci-
mens and acknowledge two anonymous reviewers for valuable sugges-
tions and comments on the manuscript. This study is part of the
University of Pavia strategic theme ‘Towards a governance model for
international migration: an interdisciplinary and diachronic perspective’
(MIGRAT-IN-G) (to A.O., A.A., O.S. and A.T.).
Disclosure statement
The authors report no conflicts of interest. The authors alone are respon-
sible for the content and writing of the paper.
Funding
This work was funded by Ministero dell'Istruzione, dell'Università e della
Ricerca [RBFR126B8I (to A.O. and A.A.)].
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